Background Many osteoarthritis (OA) patients show significant synovial involvement. Recently, we found strong upregulation of canonical Wnts 2b and 16 and WISP1, a downstream protein, in the synovium in two murine OA models. Wnt signaling has been implicated in OA through activation of the β-catenin pathway. In addition, TGF-β signaling is critical in cartilage maintenance. TGF-β signals via both ALK5 and ALK1 and downstream via Smad 2/3 and Smad 1/5/8 respectively.
Objectives Investigate the potential of canonical Wnts to skew TGF-β signaling from the protective Smad 2/3 pathway towards the Smad 1/5/8 pathway, which can induce chondrocyte hypertrophy.
Methods Pathway analysis of microarray data from the synovium of a collagenase-induced OA (CIOA) mouse model was done using DAVID software. In vivo synovial overexpression of genes from the canonical Wnt signaling pathway was achieved by intra-articular injection of adenoviral vectors. Gene expression in human chondrocytes was analyzed by qPCR. Detection of Smad 2/3 and Smad 1/5/8 phosphorylation was done by Western blot analysis. Functional TGF-β signaling via ALK5 was measured using the luciferase reporter construct CAGA-Luc.
Results Pathway analysis using DAVID, showed that both Wnt and TGF-β signaling were enriched in the synovium of mice with CIOA. Because of their small size, Wnts and WISP1 can migrate into the cartilage and possibly alter the chondrocyte phenotype. To determine if synovial overexpression of canonical Wnts leads to Wnt signaling in the cartilage, we injected adenoviral vectors for Wnt8a and Wnt16 into murine knee joints. These vectors cannot target the cartilage, due to their size. Synovial overexpression of Wnt8a and Wnt16 led to β-catenin accumulation in chondrocytes, a tell-tale sign of canonical Wnt signaling. In vitro overexpression of canonical Wnts and WISP1 in human chondrocytes led to significantly increased collagen X and decreased collagen II expression, suggesting a loss of chondrocyte phenotype. Moreover, pre-incubation with Wnt3a and/or WISP1 resulted in decreased TGF-β-induced phosphorylation of Smad 2/3, whereas phosphorylation of Smad 1/5/8 was increased in both murine and human chondrocytes. This implies a shift towards dominant TGF-β signaling via the hypertrophy-inducing ALK1 pathway. On a functional level, pre-incubation with Wnt3a and/or WISP1 led to decreased CAGA-Luc reporter construct activity, confirming decreased ALK5 signaling. Moreover, the expression of the anti-hypertrophic factor Sox9 was decreased after pre-incubation with Wnt3a and WISP1. In order to investigate whether Wnt3a skews the TGF-β signaling via the canonical signaling pathway, we pre-incubated chondrocytes with Wnt3a and/or WISP1 in the presence of DKK-1, a selective inhibitor of canonical Wnt signaling. Compared to the groups without DKK-1, we found increased Smad 2/3 phosphorylation and decreased phosphorylation of Smad 1/5/8 after Wnt3a stimulation.
Conclusions Wnts produced in the synovium may play an important role in OA pathology by changing the chondrocyte phenotype, probably through modulation of the important TGF-β signaling pathway via the canonical Wnt signaling pathway. This points towards Wnt/WISP1 expression in the synovium as an interesting target for OA therapy.
Disclosure of Interest None Declared